United States
Environmental Protection
Agency
Water Engineering
Research Laboratory
Cincinnati OH 45268
Research and Development
EPA/600/S2-87/092 Apr. 1988
SEPA Project Summary
Effect of Wastewater
Disinfectants on Survival of
R-FactorColiform Bacteria
James T. Staley, Jorge Crosa, Foppe DeWalle, and Dale Carlson
This investigation was undertaken
to assess the problem of antibiotic
resistance among total and fecal
coliform bacteria in wastewaters. Of
particular concern is the potential for
dispersal of antibiotic resistance
from enteric bacteria derived from
the human intestinal tract to other
bacteria in environments that receive
effluent from wastewater treatment
facilities. It is conceivable that
environmental strains of
opportunistic pathogens such as
Pseudomonas aeruginosa could
acquire multiple-antibiotic
resistance borne on plasmids
originating directly from enteric
bacteria. This could complicate
antibiotic therapy for patients
exposed to environmental strains of
potential pathogens.
The objectives of the study were
to determine the incidence of
antibiotic resistance among coliform
bacteria in a secondary wastewater
treatment facility and to determine
whether various alternative
disinfection procedures would select
for or against antibiotic resistant
coliform bacteria. In addition,
experiments were designed to
determine if some of the coliform
bacteria that were resistant to
several antibiotics carried their
resistance features on plasmids (R-
Factors) and, if so, to determine
whether they could transfer these
plasmids to other organisms in the
environment.
Widespread antibiotic resistance
was found among total and fecal
coliform bacteria. Levels of 20%
resistance were commonly
encountered for the various
antibiotics tested including
streptomycin, chloramphenicol,
tetracycline, and kanamycin. Higher
levels (up to 80% of total coliform
bacteria) were resistant to ampicillin,
but this resistance was associated
primarily with Klebsiella pneumonias
and thought to be chromosomal-
borne (all strains of this commonly
encountered total and fecal coliform
species that we analyzed were
resistant to ampicillin whether or not
they contained plasmids)
All wastewater treatment
procedures tested (chlorination,
ozonation, and ultraviolet
disinfection) resulted in significant
decreases both in antibiotic-
resistant as well as antibiotic-
sensitive total and fecal coliform
bacteria. No dramatic selection for
antibiotic-resistant types was
apparent during treatment although
occasionally some specific types
were found at somewhat higher
levels in secondary and disinfected
effluent than In raw wastewater. The
high diversity of total and fecal
coliform bacteria found in the
wastewaters, and the high diversity of
antibiotic-resistant patterns and
plasmid compositions among the
coliform bacteria complicated the
resolution of this question.
Attempts were made to determine
whether naturally occurring
plasmid-containing, multiple-
antibiotic resistant Escherichia coli
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could transfer resistance to
laboratory strains and vice versa.
Though this could be demonstrated
in the laboratory under conditions
simulating that of the natural
environment, the transfer rates were
low using bacterial concentrations
that were much higher than would be
normally encountered in the
environment. This suggests that
transfer may not occur in natural
environments unless there is some
unknown mechanism for
concentration of E. coli in receiving
waters.
Non-coliform heterotrophlc
bacteria from wastewaters were
found to carry equally high
proportions of antibiotic resistance.
Since these organisms occur at
about 100-fold higher con-
centrations than conform bacteria,
they may prove to be a more
important source of R-Factors for
environmental bacteria.
This Project Summary was
developed by EPA's Water Engineering
Research Laboratory, Cincinnati, OH,
to announce key findings of the
research project that is fully
documented in a separate report of
the same title (see Project Report
ordering information at back).
Introduction
For public health reasons there is
concern about the widespread dispersal
of antibiotic resistance among bacteria in
the environment. Antibiotic-resistance
traits are sometimes borne on plasmids
which can be transferred from one
species of bacterium to another. Thus,
by this mechanism, antibiotic-resistance
traits could be disseminated from enteric
bacteria of human origin through
wastewater treatment facilities to
organism in the environment that are
sensitive to the antibiotics of concern.
Some of these environmental organisms,
such as Pseudomonas aeruginosa and
Klebsiella pneumoniae, are potential
pathogens. Their acquisition of antibiotic
resistance could complicate antibiotic
therapy if they were subsequently
responsible for an infectious disease.
Wastewater is a possible major source
of antibiotic-resistance traits. Patients
receiving antibiotic therapy are known to
harbour large numbers of antibiotic-
resistant enteric bacteria in their
intestinal tracts. These enter wastewater
treatment facilities and are discharged
into receiving waters. The overall goal of
this project was to learn more about the
incidence of antibiotic resistance among
total and fecal coliform bacteria in raw
and treated wastewaters. Specific
objectives included: (a) a determination
of the extent to which antibiotic-
resistant total and fecal coliform bacteria
exist in (i) raw wastewater, (ii) treated
secondary effluent from an activated
sludge facility, and (iii) secondary
effluent disinfected by chlorination,
ozonation, and ultraviolet light; (b)
assessment of whether some of the
resistance is due to R-Factor plasmids;
and, (c) determination of whether
antibiotic resistance can be transferred
from one bacterium to another in
wastewater.
Materials and Methods
During 18 surveys of a secondary
activated sludge wastewater treatment
facility, fifty total and fecal coliform
bacterial isolates were randomly chosen
at each stage of treatment from primary
enumeration media (mEndo medium for
total coliforms and mFC medium for fecal
coliform bacteria). In early chlorination
surveys, a comparison was made
between direct selection of coliform
bacteria on the mEndo and mFC media
by incorporating antibiotics individually in
the media at 20ug/mL of each [ampicillin
(A), streptomycin (S), chloramphenicol
(C), tetracycline (T), kanamycin (K), and
naladixic acid (N)] versus indirect
selection on mEndo and mFC media
containing no antibiotics followed by
streaking for isolation on MacConkey
agar into which the antibiotics were
individually incorporated. The indirect
selection procedure in which 50
randomly chosen isolates were tested for
resistance, generally provided higher
recoveries of antibiotic-resistant strains,
so it was used in all subsequent work.
In six of the surveys, chlorination was
used in the laboratory at two
concentrations to disinfect the secondary
clarified effluent. In another six surveys,
ultraviolet light (UV) was used at two
levels as a disinfectant, and in the final
six surveys ozone was used at low and
high levels as a disinfectant.
In one survey the 50 fecal coliform
bacteria isolated from each stage of
treatment (i.e. raw wastewater, secondary
effluent and ultraviolet-treated effluent
at low and high levels of disinfection)
were identified to species using the
API-20E system (Analytab Products,
Plainview, N.Y.)* and their antibiotic
resistance determined as described
above. Strains were lysed and plasmids
were characterized using gel
electrophoresis.
The antibiotic-resistance patterns i
total heterotrophic bacteria isolated fro
standard plate count agar wer
determined in raw wastewater, secondai
clarified effluent and UV-treated effluer
Experimental Results
Direct selection procedures resulted
lower recoveries of antibiotic-resista
total and fecal coliform bacteria than wi
indirect selection (Table 1) suggestir
that coliform organisms are moi
susceptible to antibiotic effects upc
primary isolation. Thus, in all subseque
studies, indirect selection procedur<
were used to obtain isolates.
Antibiotic resistance was commc
among total and fecal coliform bacteri
The most common resistance was
ampicillin. Frequently, greater than 80
of the total coliform bacteria we
resistant to this antibiotic throughout tl
various treatment stages regardless
the disinfectant used. Generally low
levels of resistance to this antibiotic we
found among the fecal coliform isolate
This may be explained by the results
one survey (October 9, 1980), when tl
identity of all fecal coliform isolates w;
determined, that showed most strains
Escherichia coli were sensitive to th
ampicillin (Table 2). In contrast, the oth
common fecal coliform bacterium was
pneumoniae, and all of the strains of tr
organism were resistant to this antibiot
Because E. coli was the predomina
fecal coliform bacterium, the percental
of fecal coliforms resistant to A w.
reduced in comparison to total coliforr
where K. pneumoniae and related
resistant species may be predominai
Strains of K. pneumoniae that are sing
resistant to this antibiotic probably do r
carry this resistance on a plasm
because plasmids were not detected
some strains of this species and
strains were resistant to this antibio
(Table 2).
Lower levels of resistance we
usually found for antibiotics other tlr
ampicillin. However, levels of 20
resistance or greater were ofti
encountered for the other antibioti
(Table 1). Tables 3 and 4 show tl
results for another survey. Table
indicates the percentage of strai
showing resistance to each antibiotic a
Table 4 shows the antibiotic resistan
patterns of each strain from that survey
'Mention of trade names or commercial
products does not constitute endorsement or
recommendation for use
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Table 1. Comparison of Recovery of Antibiotic-Resistant Bacteria by Direct and Indirect Selection Procedures (6-13-80)
Percentage of Antibiotic-Resistant Conforms
Ampicillin Chloramphenicol Kanamycin Streptomycin
Wastewater
Direct Indirect Direct
Total Coliforms
Raw 10 80.4 0.80
Secondary 47 76.0 1.1
Low Chlorination" 5.7 87.5 9.6
High Chlorination" 6.1 81.3 0.70
Fecal Coliforms
Raw+ 1.3 48.9 0.85
Secondary 7.4 46.0 0.20
Low Chlorination 9.6 66.0 0.24
High Chlorination 19.0 60.0 0
* 48 strains checked rather than 50.
+ 47 strains checked rather than 50.
Indirect Direct
15.2 11.8
24.0 1.5
20.8 14.3
22.9 11.9
27.7 1.6
26.0 1.5
24.0 4.3
30.0 0.33
Indirect Direct Indirect
10.9 11.9 52.2
54.0 1.5 70.0
31.3 6.8 50.0
16.7 4.9 29.2
42.6 13.9 55.3
30.0 3.2 38.0
38.0 8.2 56.0
14.0 0 24.0
Tetracycline
Direct Indirect
4.5 8.7
1.7 20.0
21.1 2.1
5.2 14.6
12.2 21.3
5.3 40.0
8.6 32.0
12.7 0
Table 2. Identification and Antibiotic Resistance of Fecal Conform Bacteria from
Raw Wastewater and
Secondary Effluent from
the Renton Wastewater
Treatment Plant (October 9, 1980)
Antibiotic Resistance Pattern (No. of strains/No, of
plasmids)*
Species
Eschenchia coli
"
„
(1
Klebsiella pneumomae
Klebsiella oxytoca
Citrobacter freundii
"
Raw Wastewater
0(16/0)
0(1211-4)*
A (2!6-8); T (1!2); AT
(1/3); KS (1/2); ST
(1!3):ACS (2/1); CST
,'1);KST (1/3); AKST (1/2);
ACKST (1/5)
A (3/1 -3)
A (2/2-3)
0(2/1-6)
A (3/1-8)
Secondary Wastewater
0 (6/0)
0(1 6/1-6)
A (1/0); S (1,'3); T (1/1);
AS (2/1 -2)
A (410); A (161 1-7)
"Antibiotics tested included ampicillin (A), tetracycline (T), kanamycin (K),
streptomycin (S), and chloraphenicol (C).
+ This indicates that of these 12 strains, none were resistant to any of the
antibiotics tested and that 1 -4 plasmids were detected in each strain.
Antibiotic resistance occurred at
comparable levels among noncoliform
bacteria (Standard Plate Count isolates)
obtained from wastewater (Table 5)
Multiple resistance is also common in
this diverse group of bacteria (Table 6).
Since these are 50 to 100 times more
numerous than total coliforms and 100 to
200 times more numerous than fecal
coliforms in raw and treated wastewater,
they comprise an important reservoir for
antibiotic resistance in wastewaters.
Plasmids were responsible in part for
multiple resistance to antibiotics. For
example, during one of the UV surveys
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Table 3. Antibiotic-Resistant Coliform Indices of Renton Wastewater(August 20, 1980)
Wastewater
None
Ampic/llin
Chloramphen-
icol
Kanamycin Streptomycin Tetracycline
Total Conforms
Raw
% of unselected
Secondary
% of unselected
Secondary
chlorinated^
% of unselected
Secondary
high
chlorinated^
% of unselected
Fecal Conforms
Raw
% of unselected
Secondary
% of unselected
Secondary
chlorinated^
% of unselected
3.5x107(1.9-5.2)'
100%
4.9x105 (3.6-6.1)
100%
6.0x104
100%
3.5x102(2.3-4.6)
100%
4.5x106(3.8-5.2)
100%
2.2x104(2.1-2.3)
100%
1.5x104(0.3-1.6)
100%
1.3x107
37.0%
2.5x104
50.0%
3.7 x104
62.0%
1.8x102
52.0%
2.7X105
6.0%
1.3x103
6.0%
2.1x103
14.0%
7.0 xTO5
2.0%
ND
7.0x10°
2.0%
2.7x10s
6.0%
1.3X103
6.0%
ND
2.8x103
8.0%
2.9x104
6.0%
2.4 x103
4.0%
2.1x10'
6.0%
9.0x104
2.0%
ND
9.0x102
6.0%
4.2x106
72.0%
2.9X104
60%
9.6X103
16.0%
7.0x10'
20.0%
1.4x1Q6
30.0%
4.0x103
18.0%
3.9x103
26.0%
3.5x103
10.0%
ND
7.2x103
12.0%
2.8x10'
8.0%
9.9x105
22.0%
4.8X103
22.0%
4.2x103
28.0%
Secondary
high
chlorinated^
% of unselected
90.0(0.0-24.6) ND
100%
ND ND NQ 225
25.0%
"Numbers in parentheses indicate the 95% confidence intervals of the counts, e.g.,3.5 xio7 (1.9-5.2) would indicate that the
range was from 1.9x107 to 6.1 xlO7 with a mean of 3.5X107.
*/Vof detected, antibiotic resistance levels too low to be detected by this procedure.
fTotal chlorine dosage before and after test: low dosage (0.25-0.02 mg/L), high dosage (1.80-1.70-mg/L).
one strain of E coli (FH 14) resistant to
AKST transferred its resistance (and
plasmid) to a laboratory recipient (E coli
K12C600).
There was no consistent recurring
pattern indicating that treatment resulted
in selection of certain antibiotic-resistant
types during our surveys. However,
occasionally a particular resistance
pattern was selected for by the
disinfection process. For example, during
the UV survey of October 9, 1980, it
appeared that there was an increase in
resistance to chloramphenicol,
streptomycin and tetracycline. When the
patterns of resistance for each strain
were examined it was noted that whereas
there were no strains of fecal coliforms
resistant to AC in secondary effluent,
three were resistant following low levels
of UV and four were resistant following
high levels of UV. Likewise two strains of
fecal coliforms were resistant to ST after
low levels of UV and three were resistant
after high levels, whereas none were
resistant at the preceding secondary
stage of treatment. Resistance to AC was
found to be due almost exclusively to K.
pneumoniae; only one strain of E. coli
was found to be resistant to these two
antibiotics. In contrast, resistance to ST
was found to exclusively reside in E. coli
during this survey. E. coli strains
resistant to this antibiotic following low
levels of UV contained different plasmid
contents, although they both had a 3.2
megadalton (Md) plasmid. The three
strains of E coli resistant to ST after high
levels of UV also had plasmids. Two of
these appeared to have plasmid
compositions identical to one another;
however, they differed from the plasmid
composition of the strains of the low UV
stage. Therefore, this indicates that one
particular strain was not necessarily
being selected. However, all five ST
strains contained a plasmid of 3.2 to 3.
Md size. Thus, this particular plasmi
type may carry UV resistance as well a
resistance to ST.
Attempts were made to demonstrat
in situ conjugation in raw wastewatf
using laboratory strains and strain
selected from wastewater. Althoug
conjugation could be demonstrated, th
frequency was very low using condition
more favorable than that of actu<
wastewater. Although this does not rul
out the possibility that in situ conjugatio
might occur, it does suggest that, if
occurs, it would be uncommon in treate
wastewater.
Conclusions
Disinfection by chlorination, ultravioli
light, and ozonation is effective i
reducing not only the numbers of tot
and fecal coliform bacteria, but also th
numbers of those resistant to antibiotic
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Table 4. Number of Isolated Col/form Bacteria and Their Antibiotic Resistance Patterns from Survey
of 8/20/80 (see Table 3)
Antibiotic Resistance Pattern
Total
Raw-
Total
Secon
-darv
49'
48
Total
Cl
Total
High
Cl
Fecal
Raw
Fecal
Secon-
dary
Fecal
Cl
Fecal
High
Cl
50
50
50
50
50
73*
23
23
24
A
A
A
A
A
A
A
A
A
A
A
Total
N
K
K
K
K
C
C
N
S
S
S
S
S
S
S
S
S
S
23
29
1
1
36
1
2
1
4
33
7
17
1
12
17
Total: Total coliform bacteria obtained by standard methods on membrane filters. Fecal • Fecal conform
by bacteria obtained by standard methods on membrane filters. Raw: Raw wastewater sample, Renton.
Secondary: Secondary wastewater sample, Renton. L-UV: Low ultraviolet light dosage to secondary
wastewater
H-Uv: High ultraviolet light dosage to secondary wastewater.
* Total number of isolates obtained.
{Number of isolates with that antibiotic-resistant pattern.
"Indicates a value of < 1 coliform bacterium isolated
Thus, current treatment practices have a
major impact on reducing the total
numbers of antibiotic-resistant bacteria
that reach receiving waters. Furthermore,
there was no evidence of a major
increase in the proportion of any
antibiotic-resistant total or fecal coliform
bacteria during wastewater treatment or
disinfection. Therefore, we conclude that
current treatment and disinfection
practices are reasonably nonselective
and effective in reducing the widespread
Jispersal of antibiotic-resistant bacteria.
Total and fecal coliform bacteria
comprise only one reservoir for antibiotic
resistance among bacteria in raw
wastewater. Standard Plate Count (SPC)
bacteria occur in higher concentrations
and have similar levels of antibiotic
resistance. Therefore it would be
desirable to know the source of the
antibiotic-resistant SPC bacteria and
also determine where they acquire their
resistance. It seems reasonable that the
intestinal tract of humans carries many
antibiotic-resistant noncoliform bacteria,
and these may serve as a more
important source for these properties in
the natural environment receiving
wastewater. Thus we recommend
examining the role of noncoliform
bacteria as environmental sources of
antibiotic resistance.
It seems unlikely that E. coli is able to
transfer antibiotic resistance in raw
wastewaters based upon the results of
the conjugation experiments performed.
Their numbers are lower in raw
wastewater than expected for successful
conjugation and are reduced even further
at each successive stage of treatment
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Table 5. Antibiotic-Resistance Heterotrophic Bacteria Isolated from Renton Wastewater Treatment Facility (1/14/81)
% Antibiotic Resistant
Wastewater
Raw
Secondary
effluent (2°)
2" low UV
2" high UV
Numbers of Unselecteo"
3.4X108 (+2.8X108)*
3.2x1 07(± 2.2x1 07)
4.8X105 (±3.0x105)
1.9x1 rj3( ±1.3x103)
No. of
Isolates}
32
27
20
15
Ampi-
cillin
69
70
50
67
Chi or am -
phenicol
9
4
5
<7
%An-
tibiotic
Kana-
mycin
<3
<4
10
20
Resis-
tant
Nala-
dixic
Acid
16
4
30
40
Strep-
tomycin
12
30
5
20
Tetra-
cycline
3
4
5
27
Mean bacterial density, from three plates, per 700 mL
*95% confidence limits.
tlsolates were obtained by standard plate count procedures. Thus, they were selected from Plate Count Agar plates
incubated for 48 h at 35°C. This number refers to the number of strains.
and disinfection. Therefore, unless some
unknown and unexpected concentration
mechanism occurs, it seems doubtful
that antibiotic resistance will be
transferred commonly by enteric bacteria
in wastewater or following release of
wastewater into receiving waters. Thus,
additional treatment of wastewaters
beyond disinfection appears
unnecessary to control dispersal of
antibiotic resistance carried by enteric
bacteria.
The full report was submitted in
fulfillment of Grant No. CR807124010 by
the University of Washington under the
sponsorship of the U.S. Environmental
Protection Agency.
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Table 6. Numbers of Isolated Heterotrophic Bacteria" and Their Antibiotic Resistance Patterns
from Survey of 1114181
Antibiotic Resistance Pattern
A
C
N
S
A C
A N
A S
A T
C S
K N
N S
AC N
AC S
A K N
A NT
A S T
C S T
K N S
A K N S T
Total resistant
Raw +
(32)t
17"
1
1
1
* *
1
1
1
-
-
1
1
1
-
-
-
-
-
-
26
2° Low-UV High-UV
(27) (20) (15)
13 5 5
-
3 1
-
1
1 1
6 1
1 1
7
1 1
.
.
-
1
1
1
1
1
1
21 14 13
"Obtained by standard method procedures (see Table 5).
+Raw: Raw wastewater sample, Renton wastewater treatment facility.
2": Secondary wastewater sample, Renton wastewater treatment facility.
L-UV: Low ultraviolet light dosage to secondary wastewater.
H-UV: High ultraviolet light dosage to secondary wastewater.
f Total number of isolates obtained.
"Number of isolates with that antibiotic resistant pattern.
+ +Indicates a value of < I bacterium isolated.
&U.S. GOVERNMENT PRINTING OFFICE: 1988/548-158/67098
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James T. Staley, Jorge Cross, Foppe Dewalle, and Dale Carlson are with the
University of Washington, Seattle, WA 98195.
Albert D. Venosa is the EPA Project Officer (see below).
The complete report, entitled "Effect of Wastewater Disinfectants on Survival of
R-Factor Coliform Bacteria," (Order No. PB 88-112 339/AS; Cost $19.95,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Water Engineering Research Laboratory
U.S. Environmental Protection Agency
Cincinnati, OH 45268
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No G-35
Official Business
Penalty for Private Use $300
EPA/600/S2-87/092
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